Orthogonal site‐specific protein modification by engineering reversible thiol protection mechanisms
Orthogonal site‐specific protein modification by engineering reversible thiol protection mechanisms
复制标题
通过工程可逆硫醇保护机制进行正交位点特异性蛋白质修饰
DOI：
10.1110/ps.04965405
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发表时间：
2005
期刊：
影响因子：
8
通讯作者：
H. Hellinga
中科院分区：
文献类型：
--
作者：
J. Smith;D. Conrad;M. Cuneo;H. Hellinga
共价修饰是在蛋白质中引入新功能的一种重要策略。随着工程蛋白变得越来越复杂，通常需要以特定位点的方式引入涉及几种不同功能的多种修饰。这种正交标记方案需要对不同反应性的亲核氨基酸侧链进行独立标记。我们已经开发了两种蛋白质介导的保护方案，允许独立标记多个硫醇。这些方案利用金属配位或二硫键形成来可逆地保护Cys2His2锌指结构域中的半胱氨酸。我们用麦芽糖结合蛋白构建了这些结构域的N -和C -末端融合体，并用两种或三种不同的荧光团标记。先在MBP中反应未保护的半胱氨酸，然后去保护锌指，再反应锌指半胱氨酸，进行多次修饰。融合蛋白用两种不同的荧光基团正交标记，表现出分子内荧光共振能量转移（FRET）。这些偶联物在响应麦芽糖结合时，在发射强度上显示出三倍的比率变化。我们还证明了金属和氧化还原介导的保护方法可以结合起来产生三重独立修饰，并制备了一种具有FRET接力的三种不同荧光团标记的蛋白质。最后，用硫醇介导的固定化学方法在玻片上对标记的葡萄糖结合蛋白进行共价图案化。总之，这些实验表明，可逆硫醇保护方案提供了一种快速、直接的方法来产生多个位点特异性修饰。
Covalent modification is an important strategy for introducing new functions into proteins. As engineered proteins become more sophisticated, it is often desirable to introduce multiple, modifications involving several different functionalities in a site‐specific manner. Such orthogonal labeling schemes require independent labeling of differentially reactive nucleophilic amino acid side chains. We have developed two protein‐mediated protection schemes that permit independent labeling of multiple thiols. These schemes exploit metal coordination or disulfide bond formation to reversibly protect cysteines in a Cys2His2 zinc finger domain. We constructed a variety of N‐ and C‐terminal fusions of these domains with maltose‐binding protein, which were labeled with two or three different fluorophores. Multiple modifications were made by reacting an unprotected cysteine in MBP first, deprotecting the zinc finger, and then reacting the zinc finger cysteines. The fusion proteins were orthogonally labeled with two different fluorophores, which exhibited intramolecular fluorescene resonance energy transfer (FRET). These conjugates showed up to a threefold ratiometric change in emission intensities in response to maltose binding. We also demonstrated that the metal‐ and redox‐mediated protection methods can be combined to produce triple independent modifications, and prepared a protein labeled with three different fluorophores that exhibited a FRET relay. Finally, labeled glucose‐binding protein was covalently patterned on glass slides using thiol‐mediated immobilization chemistries. Together, these experiments demonstrated that reversible thiol protection schemes provide a rapid, straightforward method for producing multiple, site‐specific modifications.
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DOI：
10.1073/pnas.96.3.893
发表时间：
1999-02-02
影响因子：
11.1
作者：
Ha, TJ;Ting, AY;Weiss, S
通讯作者：
Weiss, S
影响因子：
15
作者：
H. Watrob;C. Pan;M. Barkley
通讯作者：
H. Watrob;C. Pan;M. Barkley
影响因子：
56.9
作者：
SHI, YG;BERG, JM
通讯作者：
BERG, JM
影响因子：
2.9
作者：
Zhang, ZW;Smith, BAC;Schultz, PG
通讯作者：
Schultz, PG
DOI：
10.1073/pnas.94.9.4366
发表时间：
1997-04-29
影响因子：
11.1
作者：
Marvin, JS;Corcoran, EE;Hellinga, HW
通讯作者：
Hellinga, HW